Journal of Human Genetics (2020) 65:35–40 https://doi.org/10.1038/s10038-019-0675-4 REVIEW ARTICLE Portable sequencer in the fight against infectious disease 1 1 2 Arthur Elia Mongan ● Josef Sem Berth Tuda ● Lucky Ronald Runtuwene Received: 4 June 2019 / Revised: 11 September 2019 / Accepted: 13 September 2019 / Published online: 3 October 2019 © The Author(s) 2019. This article is published with open access Abstract Infectious disease is still a major threat in the world today. Five decades ago, it was considered soon to be eradicated, but the adaptation of pathogens to environmental pressure, such as antimicrobials, encouraged the emergence and reemergence of infectious disease. The fight with infectious disease starts with prevention, diagnosis, and treatment. Diagnosis can be upheld by observing the cause of disease under the microscope or detecting the presence of nucleic acid and proteins of the pathogens. The molecular techniques span from classical polymerase chain reaction (PCR) to sequencing the nucleic acid composition. Here, we are reviewing the works have been undertaken to utilize a portable sequencer, MinION, in various aspects of infectious disease management. 1234567890();,: 1234567890();,: Infectious disease has always been intertwined with human insecticide, is one of the factors that prevent the eradication history. Poliomyelitis was already documented in Egyptian of malaria worldwide in 1955–1978 [3]. Other example is papyrus. Leprosy, plague, cholera, yellow fever, influenza the human immunodeficiency virus emergence in the past epidemics, or pandemics were the norm [1]. As the civili- century due to simian immunodeficiency virus coincidental zation went forward, the development of microscope transmission to humans [4–6] and the 2009 H1N1 pandemic allowed the visualization of microorganisms for the first influenza virus that emerged from pigs, is a legacy of time, changing the paradigm of infectious disease [2]. Soon, influenza A virus causing pandemic in 1918 [7]. the discovery of pathogenic agents displaced the preexisting The definitive diagnosis of many infectious diseases still theories of the cause of infectious disease. Vaccines were relies on direct observation of the causing pathogens. developed to prevent contracting or spreading the disease However, at many times this is difficult to achieve, espe- and methods of food sterilization were deemed valuable for cially for viral diseases because of the size of virus particles. disease control. For example, the microscopic examination of acid-fast The fight against infectious agents was so efficient that in bacilli remains the main tool for tuberculosis detection, yet the 1960s, control and prevention measures had decreased the technique sensitivity varies on smearing, staining, and the incidence of many infectious diseases [1]. Smallpox and slide reading [8]. Parasites such as microfilaria depends on rinderpest were controlled from the world. Nevertheless, the sampling time for positive observation [9]. Therefore, emerging and reemerging infections are becoming sig- molecular techniques, such as PCR or enzyme-linked nificant worldwide problems. Pathogen adaptation seems to immunosorbent assay have been developed to assist in a hold the key for the emergence or reemergence of infectious more reliable diagnosis. They are designed to detect the disease. The acquisition of drug resistance, for example presence of infecting pathogen molecularly. Because it malaria parasites to chloroquine or the mosquito vector to directly measures the nucleic acids or proteins of infecting pathogens, the risk of missing the organism in direct observation may be avoided. Traditionally, laboratory test for drug resistance is a long * Lucky Ronald Runtuwene and laborious process. First, culturable microorganisms [email protected] must be cultured on various growth mediums. The time [email protected] needed for colonies to form also varies greatly. Then the 1 Faculty of Medicine, Sam Ratulangi University, colonies must be subjected to different type of anti- Manado, Indonesia microbials until the susceptibility or resistance can be dis- 2 Graduate School of Frontier Sciences, The University of Tokyo, tinguished. In certain cases, drug resistance analysis is very Kashiwa, Japan critical to patients’ prognosis, therefore traditional process 36 A. E. Mongan et al. terminating nucleotides to a DNA polymerization process [14]. These termination nucleotides can be paired with excitable compounds that is excited by laser beam; and depending on the compound, the excitation process will emit different wavelengths which can be identified as dif- ferent nucleotide. A different kind of DNA sequencer uses nanopore protein to recognize the nucleotide in question. Here, the disturbance in the basal electrical current inside the protein caused by certain nucleotide strands will be recognized as specific patterns and converted into sequences of DNA by specific algorithm [16]. The deviation from common sequencing principles allows the new sequencing platform to be miniaturized Fig. 1 The portable sequencer, MinION, is being handled prior to (Fig. 1). This opens new powerful aspects of sequencing sequencing that could never been achieved before. The portable sequencer, MinION, can be transported to countries or locations where performing sequencing is difficult or is impractical. Here is the niche for genetic analysis to transferring samples to other countries or locations is for- expedite the drug resistance tests. bidden. For example, MinION has been taken to a remote There are many genetic analyses designed to confirm rainforest of Tanzania [17], Ecuador [18], the Canadian diagnosis or to test for microbial resistance. PCR can be high Arctic [19], and even the International Space Station used to detect the presence of pathogen DNA. Using mul- [20]. MinION helped the surveillance and sequencing of tiplex primers targeting DNA of many organisms, an Zika virus in the 2016 Brazil outbreak [21]. During the investigator can detect the presence of single or multiple project, it was found that most of the genome were frag- infecting agents [10]. Further investigation using primers mentary. Low titer was correlated to sequencing less than targeting the presence of known mobile genetic elements 50% coverage, so ‘tiling amplification’ was used to amplify can decipher the antibiotics resistance that may be present in the whole genome of Zika virus. This technique also was the clinical sample [11]. PCR can also be used to check for employed in the Ebola virus (EBOV) outbreak in Guinea to single nucleotide polymorphisms conferring drug resis- get a high nucleic acid concentration for sequencing [22]. tance. By creating primers matched the substituted nucleo- The portability of MinION comes at the expense of tide, the susceptibility or resistance can be determined by sequencing accuracy. Despite recent improvements to the the absence or presence of certain bands [12]. Combination chemistry and computational tools, the sequencing errors with real-time PCR using probes specific to mutations will are still between 5 and 15% [23]. Consensus calling using amplify mutated gene and can be observed in real time [13]. bioinformatics tools can improve accuracy to more than Sequencing of PCR amplicons or whole pathogen 97% [24, 25]. When consensus calling was applied to genomic DNA can be applied to comprehensively screen simultaneously screen for multiple antimalarials resistance, for infecting agents and their drug resistance phenotype, if it was clear that north Indonesia, north Vietnam, and exists, at the same time. Sequencing technique has been southeast Thailand had different mutation patterns in K13 around since 1975 by the works of Frederick Sanger, Allan gene [26], the gene related to artemisinin resistance, that Maxam, and Walter Gilbert [14]. Since then, the technology were in concordance with a major paper describing the has been modified and refined with the automation of the artemisinin-resistance related mutations around the Mekong original technique, the inception of next-generation region [27]. Nevertheless, the mutations conferring resis- sequencing, and the advent of long-read sequencing. With tance to chloroquine were similar in these three regions. each iteration, the data throughput is increased, cost is This method applies the amplification of multiple genes reduced, and physical form of the sequencer is reduced. with PCR. The sequencing depth is high so consensus Sequencing ensures detection of DNA composition unique sequence can be generated with high confidence. Targeted to an organism, the presence of antimicrobials-destroying sequencing is cost efficient by performing multiplex genes, or mutation that can change the function or con- sequencing. Although the running cost of other sequencing formation of proteins related to drug resistance. Further platform is cheaper than MinION (for example, $67.82 for application of sequencing using the next-generation plat- MiSeq compared with $71.56 for MinION per sample [28]), forms encompasses epigenetic profiling as well [15]. MinION has significantly lower cost to set up. Next-generation sequencing incorporates many technol- Targeted sequencing with MinION is powerful and fast ogies, but mainly they include the incorporation of to detect pathogens in clinical samples. Bacterial Portable sequencer in the fight against infectious disease 37 Table 1 An outline of MinION MinION sequencing method Techniques applied Reference sequencing methods and